Cystic fibrosis (CF) is the most common fatal genetic disease in humans (Boat et al., 1989). Based on both genetic and molecular analysis, a gene associated with CF was recently isolated as part of 21 individual cDNA clones and its protein product predicted (Kerem et al., 1989; Riordan et al., 1989; Rommens et al., 1989). U.S. Ser. No. 488,307 describes the construction of the gene into a continuous strand and confirmed the gene is responsible for CF by introduction of a cDNA copy of the coding sequence into epithelial cells from CF patients (See also Gregory et al., 1990; Rich et al., 1990). Wild type but not a mutant version of the cDNA complemented the defect in the cAMP regulated chloride channel shown previously to be characteristic of CF. Similar conclusions were reported by others (Drumm et al., 1990).
The protein product of the CF associated gene is called the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al., 1989). CFTR is a protein of approximately 1480 amino acids made up of two repeated elements, each comprising six transmembrane segments and a nucleotide binding domain. The two repeats are separated by a large, polar, so-called R-domain containing multiple potential phosphorylation sites. Based on its predicted domain structure, CFTR is a member of a class of related proteins which includes the multi-drug resistance (MDR) or P-glycoprotein, bovine adenyl cyclase, the yeast STE6 protein as well as several bacterial amino acid transport proteins (Riordan et al., 1989; Hyde et al., 1990). Proteins in this group, characteristically, are involved in pumping molecules into or out of cells.
CFTR is a large, multi domain, integral membrane protein which is postulated to regulate the outward flow of anions from epithelial cells in response to phosphorylation by cyclic AMP-dependant protein kinase or protein kinase C (Riordan et al., 1989; Welsh,1986; Frizzel etal., 1986; Welsh and Liedtke, 1986; Schoumacher et al., 1987; Li et al., 1988; Hwang et al., 1989; Li et al., 1989).
To investigate the function of the CFTR, the mechanism by which mutations in the CFTR gene cause cystic fibrosis, to develop potential therapies for cystic fibrosis, and for many other applications, a cDNA clone encoding the entire CFTR protein is necessary.
It is an aspect of the present invention to engineer CFTR cDNA sequences containing all of the coding information for CFTR protein on a single recombinant DNA molecule which can be stably propagated in E. coli and transferred to yeast, insect, plant or mammalian cells, or transgenic animals, for expression of wild-type CFTR protein, as well as mutants to provide derivatives which correlate with the cystic fibrosis disease.
It is another aspect to provide the critical cDNA gene containing the correct gene sequence in order to provide for production of the CFTR protein.
It is yet another aspect to enable various diagnostic, therapeutic and protein production techniques related to the evaluation and treatment of cystic fibrosis caused by faulty CFTR function, faulty CFTR processing or related to the intracellular location of CFTR.
In addition, a mutation within the gene sequence encoding CFTR protein has been identified in DNA samples from patients with cystic fibrosis, the most common genetic disease of caucasians (Kerem et al., 1989). The mutation, which results in the deletion of the amino acid phenylalanine at position 508 of the CFTR amino acid sequence, is associated with approximately 70% of the cases of cystic fibrosis.
This mutation in the CFTR gene results in the failure of an epithelial cell chloride channel to respond to cAMP (Frizzell et al., 1986; Welsh, 1986; Li et al., 1988; Quinton, 1989). In airway cells, this leads to an imbalance in ion and fluid transport. It is widely believed that this causes abnormal mucus secretion, and ultimately results in pulmonary infection and epithelial cell damage. That the chloride channel can be regulated by cAMP in isolated membrane patches (Li et al., 1988) suggests that at least some CFTR is present in the apical plasma membrane and that CFTR responds to protein kinase A. Whether CFTR itself is a regulator of the membrane chloride channel or constitutes the channel itself remains controversial.
U.S. Ser. No. 488,307, fully incorporated herein, showed that CFTR is a membrane-associated glycoprotein that can be phosphorylated in vitro (Gregory et al., 1990). The protein has a primary translation product which migrates with apparent molecular weight on SDS-polyacrylamide gels of 130k (referred to as band A). In vaccinia virus-infected, cDNA transfected HeLa cells or in reticulocyte lysates containing canine pancreatic membranes, band A is modified by glycosylation to yield a version of apparent molecular weight 135 kd called band B. The use of polyclonal and monoclonal antibodies to CFTR showed that non-recombinant T84 cells contain, in addition, a diffusely migrating 150 kd (band C) version of CFTR.
It is another aspect of the present invention to study structure:function relationships in CFTR by constructing a site specific mutation which provides for the deletion of phenylalanine 508 (referred to as .DELTA.F508).
It is yet another aspect to characterize variant CFTR protein forms associated with a number of less frequent CF associated mutations, as well as mutations in residues predicted to play an important role in the function of CFTR.
It is still yet another aspect of the present invention to more fully describe the characteristics of CFTR associated with bands a, b and c.
It is yet still another aspect of the present invention to provide new diagnostic and therapeutic methods for CF which rely upon intracellular processing mechanism for CFTR and intracellular location of variously processed CFTR.